Ultra high affinity neutralizing antibodies

ABSTRACT

Ultra high affinity antibodies with binding affinities in the range of 10 10  M −1 , and even 10 11  M −1  are disclosed. Such antibodies include antibodies having novel high affinity complementarity determining regions (CDRs), especially those with framework and constant regions derived from either humans or mice. Methods of preparing and screening such antibodies, as well as methods of using them to prevent and/or treat disease, especially virus-induced diseases, are also disclosed.

This application claims priority of U.S. Provisional Application Ser.No. 60/178,426, filed 27 Jan. 2000, the disclosure of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel ultra high affinity neutralizingantibodies.

The current incidence of infection caused by resistant or difficult tocontrol microbes has created a need for newer approaches to controllingsuch organisms, as well as to treating those already infected.

Among the more difficult infectious agents to control and treat are theviruses. For example, respiratory syncytial virus (RSV) is the majorcause of acute respiratory illness in young children admitted tohospitals and the major cause of lower respiratory tract infection inyoung children. A major obstacle to producing an effective vaccineagainst such agents as RSV has been the issue of safety. Conversely, theuse of immunoglobulins against such viral agents has proven of somevalue. For example, studies have shown that high-titred RSVimmunoglobulin was effective both in prophylaxis and therapy for RSVinfections in animal models.

An alternative approach has been the development of antibodies,especially neutralizing monoclonal antibodies, with high specificneutralizing activity. One drawback to this route has been the need toproduce human antibodies rather than those of mouse or rat and thusminimize the development of human anti-mouse or anti-rat antibodyresponses, which potentially results in further immune pathology.

An alternative approach has been the production of human-murine chimericantibodies in which the genes encoding the mouse heavy and light chainvariable regions have been coupled to the genes for human heavy andlight chain constant regions to produce chimeric, or hybrid, antibodies.

In some cases, mouse CDRs have been grafted onto human constant andframework regions with some of the mouse framework amino acids beingsubstituted for correspondingly positioned human amino acids to providea “humanized” antibody. [Queen, U.S. Pat. Nos. 5,693,761 and 5,693,762].However, such antibodies contain intact mouse CDR regions and have metwith mixed effectiveness, producing affinities often no higher than 10⁷to 10⁸ M⁻¹.

A humanized anti-RSV antibody with good affinity has been prepared andis currently being marketed. [See: Johnson, U.S. Pat. No. 5,824,307]

The production of ultra high affinity antibodies would be desirable fromthe point of view of both the neutralizing ability of such an antibodyas well as from the more practical aspects of needing to produce lessantibody in order to achieve a desirable degree of clinicaleffectiveness, thereby cutting costs of production and/or allowing agreater degree of clinical effectiveness for administration in the samevolume.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to high affinity neutralizing antibodiesand active fragments thereof exhibiting affinity constants of at least10¹⁰ M⁻¹, and even 10¹¹ M⁻¹, and more specifically to such aneutralizing monoclonal immunoglobulin, including antibodies and/oractive fragments thereof, wherein the antibody and/or fragment has humanconstant regions.

The present invention solves the above-mentioned problems by providinghigh affinity neutralizing antibodies without the presence of intactmouse CDR regions that cause human anti-mouse antibody reactions (HAMA)and with sufficiently high affinity neutralizing activity to reduce costand efficacy of overall production.

One aspect of the present invention relates to high affinityneutralizing antibodies with affinity constants of at least 10¹⁰ M⁻¹,and even 10¹¹ M⁻¹, and with specificity towards specific antigenicdeterminants, such as those exhibited by virus-expressed proteins.

One object of the present invention is to provide such high affinityneutralizing antibodies with specificity toward antigens produced byviruses, such as where such antigens are expressed by virus-infectedcells in a mammal, especially a human.

In one such embodiment, the high affinity neutralizing immunoglobulin,including antibodies and/or active fragments thereof, of the presentinvention, and active fragments thereof, are specific for respiratorysyncytial virus (RSV), most especially for the F antigen expressed bysaid RSV and also expressed on the surfaces of cells infected with RSV(the presence of which antigen on the cell surface causes fusion of thecells into syncytia),

Thus, in one embodiment, a high affinity neutralizing immunoglobulin,including antibodies and/or active fragments thereof, of the presentinvention binds to the same epitope on RSV as the antibody whose lightchain variable chain has the sequence of SEQ ID NO: 1 (shown in FIG. 1A)and whose heavy chain variable chain has the sequence of SEQ ID NO: 2(shown in FIG. 1B).

It is an object of the present invention to provide ultra high affinityneutralizing antibodies having substantially the framework regions ofthe immunoglobulin disclosed in FIG. 1 (with the same specificity ofthat immunoglobulin, which is an anti-RSV antibody structure) butwherein the immunoglobulins, including antibodies and active fragmentsthereof, of the present invention contain one or more CDRs(complementarity determining regions) whose amino acid sequences areindependent of those in the so-called reference antibody, although saidsequences may, in some cases, differ by no more than one amino acid andthis may be limited to a difference in only one of said CDR regions.

In a preferred embodiment of the present invention, the novelimmunoglobulins of the present invention will differ from the antibodyof FIG. 1 (hereafter, the “basic antibody” or “reference antibody” or“reference immunoglobulin”) only in the sequences of one or more of theCDRs and, in a most preferred embodiment these differences occur only inCDRs L2, L3, H1, and H3.

Especially preferred embodiments of the present invention have theframework sequences depicted in FIG. 1, thus having the heavy and lightchain variable sequences depicted in FIGS. 3, 4, 5, 6, and 7.

In one embodiment, the high affinity neutralizing antibodies of theinvention include a human constant region.

In a preferred embodiment, a high affinity RSV-neutralizing antibody ofthe invention, including active fragments thereof, with an affinityconstant (K_(a)) of at least as high as 10¹⁰ M⁻¹, and even 10¹¹ M⁻¹, isa recombinant immunoglobulin, such as an antibody or active fragmentthereof, that includes a human constant region and framework regions forthe heavy and light chains wherein at least a portion of the frameworkis derived from a human antibody (or from a consensus sequence of ahuman antibody framework), an example of said framework regions depictedfor the antibody sequences of FIG. 1.

In one embodiment, all of the framework is derived from a human antibody(or a human consensus sequence).

In another highly specific embodiment, a high affinity RSV-neutralizingantibody, with an affinity of at least 10¹⁰ M⁻¹, is a recombinantantibody having a human constant region, one or more CDRs that arederived from a non-human antibody in which at least one of the aminoacids in at least one of the CDRs is changed and in which all or aportion of the framework is derived from a human antibody (or aconsensus sequence of a human antibody framework).

In a separate embodiment, a humanized neutralizing immunoglobulin thatbinds to the same epitope as the basic or reference antibody orimmunoglobulin whose variable chains are shown in FIG. 1, and that hasan affinity of at least 10¹¹ M⁻¹, includes at least one of the followingamino acids at the following positions of the CDRs: an alanine atposition 2 of CDR H1, a phenylalanine at position 6 of CDR H3, aphenylalanine at position 3 of CDR L2, and a phenylalanine at position 5of CDR L3. Other embodiments comprise other single amino acidsubstitutions at these locations.

It is another object of the present invention to provide compositionscomprising the immunoglobulins disclosed herein wherein said structuresare suspended in a pharmacologically acceptable diluent or excipient.

It is a still further object of the present invention to provide methodsof preventing and/or treating respiratory syncytial virus comprising theadministering to a patient at risk thereof, or afflicted therewith, of atherapeutically effective amount of a composition containing animmunoglobulin of the invention, such as where said antibodies or activefragments thereof exhibit the specificity and affinity propertiesdisclosed herein for the immunoglobulins of the invention.

DEFINITIONS

The term “antigen” refers to a structure, often a polypeptide orprotein, present on the surface of a microorganism, such as a virus, forwhich an antibody has affinity and specificity.

The term “antigenic determinant” refers to a specific binding site on anantigenic structure for which an immunoglobulin, such as an antibody,has specificity and affinity. Thus, a particle, such as a virus, mayrepresent an antigen but may have on its surface a number of separate,and different, antigenic sites such as where the virus has a number ofdifferent surface proteins and each represents a distinct potentialbinding site for an immunoglobulin.

The term “immunoglobulin” refers to a protein or polypeptide havingspecificity and affinity for an antigen or antigenic determinant. Thisterm includes antibodies, commonly depicted as tetrameric, as well asactive fragments thereof, such fragments having specificity and affinityfor antigens or antigenic determinants. Thus, “immunoglobulin” as usedherein includes antibodies and all active fragments thereof.

The term “antibody” refers to a protein or polypeptide having affinityfor an antigenic determinant, usually one found on the surface of amicroorganism, especially a virus. Such an antibody is commonly composedof 4 chains and is thus tetrameric.

The term “neutralizing immunoglobulin” or “neutralizing antibody” refersto the ability of the immunoglobulins, including antibodies, of thepresent invention to reduce the replication of microorganisms,especially viruses, in organisms as well as in cell cultures. Anindication of such ability is the data from the microneutralizationassays disclosed hereinbelow. Such a structure usually has both variableand constant regions whereby the variable regions are mostly responsiblefor determining the specificity of the antibody and will comprisecomplementarity determining regions (CDRs).

The term “complementarity determining region” or “CDR” refers tovariable regions of either H (heavy) or L (light) chains contains theamino acid sequences capable of specifically binding to antigenictargets. These CDR regions account for the basic specificity of theantibody for a particular antigenic determinant structure. Such regionsare also referred to as “hypervariable regions.”

The term “active fragment” refers to a portion of an antibody that byitself has high affinity for an antigenic determinant and contains oneor more CDRs accounting for such specificity. Non-limiting examplesinclude Fab, F(ab)′₂, heavy-light chain dimers, and single chainstructures, such as a complete light chain or complete heavy chain.

The term “specificity” refers to the ability of an antibody to bindpreferentially to one antigenic site versus a different antigenic siteand does not necessarily imply high affinity.

The term “affinity” refers to the degree to which an antibody binds toan antigen so as to shift the equilibrium of antigen and antibody towardthe presence of a complex formed by their binding. Thus, where anantigen and antibody are combined in relatively equal concentration, anantibody of high affinity will bind to the available antigen so as toshift the equilibrium toward high concentration of the resultingcomplex.

The term “affinity constant” refers to an association constant used tomeasure the affinity of an antibody for an antigen. The higher theaffinity constant the greater the affinity of the immunoglobulin for theantigen or antigenic determinant and vice versa. An affinity constant isa binding constant in units of reciprocal molarity. Such a constant isreadily calculated from the rate constants for theassociation-dissociation reactions as measured by standard kineticmethodology for antibody reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of the light and heavy chainvariable regions of an anti-RSV antibody wherein the CDR regions areunderlined while non-underlined residues form the framework regions ofthe variable regions of each chain. In this antibody, the CDRs arederived from a mouse anti-RSV antibody while the framework regionsconsist mostly of sequences derived from a human antibody. For each CDR,locations at which amino acid replacements were used to achieve the highaffinity CDRs and antibodies disclosed herein are in bold face. Inaccordance with the disclosure herein, such replacements were only inCDRs L2, L3, H1 and H3. FIG. 1A shows the light chain variable regionand FIG. 1B shows the heavy chain variable region of the light and heavychains, respectively. Constant region sequences are not shown. Thesesequences are present in the basic clone (see Table 2), designatedIX-493 throughout this disclosure (i.e., SWSG—meaning a serine (S) atthe key position (see tables 1 and 3) of high affinity CDR H1, atryptophan (W) at the key position of high affinity CDR H3, a serine (S)at the key position of high affinity CDR L2, and a glycine (G) at thekey position of high affinity CDR L3). For purposes of this disclosure,this is the “reference antibody.”

FIG. 2 shows affinity comparisons for a particular set of beneficial orhigh affinity clones. The clonal designations are on the left of thelegend at the right of the drawing along with the indicatedsubstitutions at CDRs H1, H3, L2, and L3 shown on the right of thelegend. Measurements are by ELISA (OD at 560 nm shown on the left axis).Clone L1FR represents the results for the reference antibody structureof FIG. 1.

FIG. 3 shows the heavy and light chain variable regions for thepreferred embodiment of clone 1 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

FIG. 4 shows the heavy and light chain variable regions for thepreferred embodiment of clone 2 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

FIG. 5 shows the heavy and light chain variable regions for thepreferred embodiment of clone 3 (Table 2) of the invention disclosedherein. CDR regions are underlined while the amino acid differencesversus the antibody of FIG. 1 are indicated in bold face. Thus, thispreferred (i.e., high affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹⁰ M⁻¹) than the basic or reference antibody.

FIG. 6 shows the heavy and light chain variable regions for the mostpreferred embodiment of clone 22 (Table 4) of the invention disclosedherein. CDR regions are underlined while the amino acid changes versusthe antibody of FIG. 1 are indicated in bold face. Thus, this mostpreferred (i.e., highest affinity) antibody has several of the highaffinity CDRs (Table 3) present which give rise to higher affinity (over10¹¹ M⁻¹) than the basic or reference antibody).

FIG. 7 shows the heavy and light chain variable regions for thepreferred embodiment of clone 23 (Table 4) of the invention disclosedherein. CDR regions are underlined while the amino acid changes versusthe antibody of FIG. 1 are indicated in bold face. Thus, this preferred(i.e., highest affinity antibody, has several of the high affinity CDRs(Table 3) present which give rise to higher affinity (over 10¹¹ M⁻¹)than the basic or reference antibody).

FIG. 8 shows the results of microneutralization experiments on severalof the ultra-high affinity antibody clones disclosed herein. The aminoacids present at the key positions of the high affinity complementaritydetermining regions (see Table 3) are shown at the right in the orderH1, H3, L2, and L3 (as also shown in Table 2 where the clones are simplynumbered but the table compares to this figure by relying on the actualamino acids used at the critical positions as disclosed in the Table andin the legend at the right of this figure). Thus, for clone 4D2-7, thereis an alanine (A) at the key position of high affinity CDR H1, aphenylalanine (F) at the key position of high affinity CDR H3, aphenylalanine (F) at the key position of high affinity CDR L2, and aphenylalanine (F) at the key position of high affinity CDR L3. Briefly,about 25,000 HEp-2 cells were added to each of the wells of a 96 wellplate along with RSV and a given concentration of the antibody (antibodyconcentration per well is shown on the abscissa—See Example 2 for exactdetails). After 5 days growth, the cells were fixed, treated withbiotinylated anti-F MAb, then bound to avidin-peroxidase complex and theability of the bound peroxidase to react thionitrobenzoic acid wasdetermined by measuring O.D. at 450 nm. The amount of F protein presentwas an indicator of the extent of viral replication thereby resulting inmore reaction of substrate by peroxidase and increased absorption. Thus,the lower the OD 450 value, the greater the neutralizing ability of theindicated concentration of the given antibody. Here, IX-493(SWSG—meaning a serine (S) at the key position of high affinity CDR H1,a tryptophan (W) at the key position of high affinity CDR H3, a serine(S) at the key position of high affinity CDR L2, and a glycine (G) atthe key position of high affinity CDR L3) is the “reference antibody”(also denoted L1FR and IX493L1FR).

FIG. 9 shows results for microneutralization assays of several of theantibodies disclosed herein but where only Fab fragments were employedfor neutralization of antibody replication. Here, IX-493 (SWSG) is thereference Fab fragment and is derived from antibody Medi-493 (see:Johnson et al (1997)-ref. 23).

FIG. 10 shows the results of microneutralization for an antibodyspecific for RSV as compared to similar experiments for Fab fragments ofthe same antibody. Here, Medi 493 represents the antibody while IX-493LIFR represents the Fab fragment of this antibody. The other lines arefor Fab fragments of several of the antibodies produced according to thepresent invention (given various letter-digit code designations forinternal identification but having nothing to do with their relativeefficacy as neutralizing antibodies).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ultra high affinity neutralizingantibodies and active fragments thereof having affinity constants of atleast 10¹⁰ M⁻¹. Active fragments of these antibodies are fragmentscontaining at least one high affinity complementarity determining region(CDR).

With the advent of methods of molecular biology and recombinanttechnology, it is now possible to produce antibody molecules byrecombinant means and thereby generate gene sequences that code forspecific amino acid sequences found in the polypeptide structure of theantibodies. Such antibodies can be produced by either cloning the genesequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with in vitro assembly ofthe synthesized chains to form active tetrameric (H₂L₂) structures withaffinity for specific epitopes and antigenic determinants. This haspermitted the ready production of antibodies having sequencescharacteristic of neutralizing antibodies from different species andsources.

Regardless of the source of the immunoglobulins, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all immunoglobulins have a similar overall 3 dimensionalstructure. In the case of an antibody, this structure is often given asH₂L₂ and refers to the fact that antibodies commonly comprise 2 light(L) amino acid chains and 2 heavy (H) amino acid chains. Both chainshave regions capable of interacting with a structurally complementaryantigenic target. The regions interacting with the target are referredto as “variable” or “V” regions and are characterized by differences inamino acid sequence from antibodies of different antigenic specificity.

The variable region of either H or L chains contains the amino acidsequences capable of specifically binding to antigenic targets. Withinthese sequences are smaller sequences dubbed “hypervariable” because oftheir extreme variability between antibodies or active fragments ofdiffering specificity. Such hypervariable regions are also referred toas “complementarity determining regions” or “CDR” regions. These CDRregions account for the basic specificity of the antibody for aparticular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all canonical antibodies each have 3 CDR regions, eachnon-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for therespective light (L) and heavy (H) chains. The accepted CDR regions havebeen described by Kabat et al, J. Biol. Chem. 252:6609-6616 (1977). Thenumbering scheme is shown in the figures, where the CDRs are underlinedand the numbers follow the Kabat scheme.

In all mammalian species, antibody polypeptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

The immunoglobulins disclosed according to the present invention affordextremely high affinity (on the order of 10¹⁰ M⁻¹, and even 10¹¹ M⁻¹,for the affinity constant, or K_(a), defined as an association constantdescribing the binding of antibody and antigen as the ligands) forepitopes, or antigenic determinants, found in macromolecules, especiallyproteins, and most especially proteins expressed by viruses and othermicroorganisms, such as proteins expressed on the surfaces of viruses aswell as the surfaces of cells infected with a virus. The high affinityantibodies of the present invention are neutralizing antibodies and thusreduce the replication of viruses in organisms as well as in cellcultures while maintaining sufficient homology to the antibody aminoacid sequences of the recipient so as to prevent adverse immunologicalpathology. The latter feature is achieved through the use of constantregions similar to those of the recipient organism, such as a mammal andmost especially a human. This feature is also achieved through the useof framework amino acid sequences similar, if not identical, to thosefound in antibodies from the recipient organism.

In the latter case, some amino acid replacements may be made in theframework sequences so as to facilitate and maintain the high affinityinteraction between the novel CDRs of the present invention and theantigen for which said antibodies show specificity.

As used herein, terms such as “antibody” and “active fragment” or“fragment” are not to be considered limiting in determining the fullextent of the present invention. Thus, the fact that the term “antibody”is used rather than “active fragment” or “immunoglobulin” is not to betaken as a limitation on the invention or its uses so long as therequisite properties of specificity and affinity are exhibited by saidstructure.

In accordance with the invention disclosed herein, the affinityconstants characterizing the affinities of the high affinityneutralizing antibodies, and active fragments thereof, of the presentinvention are association constants and were measured by the kinetics ofantigen-antibody complex formation, with the rate constant forassociation to form the complex being denoted as k_(assoc) or k_(on) andthe dissociation constant being denoted as k_(diss) or k_(off).Measurement of such constants is well within the ordinary skill in theart and the details will not be described further herein except forgeneral methodology and specific conditions, where appropriate, asrecited in the examples given herein to further describe the invention.

The high affinity antibodies of the present invention can be achieved,as already described, through genetically engineering appropriateantibody gene sequences, i.e., amino acid sequences, by arranging theappropriate nucleotide sequences and expressing these in a suitable cellline. Any desired nucleotide sequences can be produced using the methodof codon based mutagenesis, as described, for example, in U.S. Pat. Nos.5,264,563 and 5,523,388. Such procedures permit the production of anyand all frequencies of amino acid residues at any desired codonpositions within an oligonucleotide. This can include completely randomsubstitutions of any of the 20 amino acids at an desired position or inany specific subset of these. Alternatively, this process can be carriedout so as to achieve a particular amino acid a desired location withinan amino acid chain, such as the novel CDR sequences according to theinvention. In sum, the appropriate nucleotide sequence to express anyamino acid sequence desired can be readily achieved and using suchprocedures the novel CDR sequences of the present invention can bereproduced. This results in the ability to synthesize polypeptides, suchas antibodies, with any desired amino acid sequences. For example, it isnow possible to determine the amino acid sequences of any desireddomains of an antibody of choice and, optionally, to prepare homologouschains with one or more amino acids replaced by other desired aminoacids, so as to give a range of substituted analogs.

In applying such methods, it is to be appreciated that due to thedegeneracy of the genetic code, such methods as random oligonucleotidesynthesis and partial degenerate oligonucleotide synthesis willincorporate redundancies for codons specifying a particular amino acidresidue at a particular position, although such methods can be used toprovide a master set of all possible amino acid sequences and screenthese for optimal function as antibody structures or for other purposes.Such methods are described in Cwirla et al, Proc. Natl. Acad. Sci.87:6378-6382 (1990) and Devlin et al., Science 249:404-406 (1990).

In accordance with the invention disclosed herein, enhanced antibodyvariants can be generated by combining in a single polypeptide structureone, two or more novel CDR sequences according to the invention, eachshown to independently result in enhanced binding activity. In thismanner, several novel amino acids sequences can be combined into oneantibody, in the same or different CDRs, to produce high affinityantibodies within the present invention. For example, 3 such novel CDRsequences may be employed and the resulting antibodies screened foraffinity for a particular antigenic structure, such as the F antigen orRSV. The overall result would thus be an iterative process of combiningvarious single amino acid substitutions and screening the resultingantibodies for antigenic affinity in a step by step manner. Such methodswere used to prepare some of the antibodies embodied within the presentinvention. Such methods also represent a convenient, if tedious, meansof optimizing the antibody sequences of the present invention,especially the sequences of the CDR regions of said antibodies.

Using the novel sequences and methods of the present invention, such anapproach avoids the time and expense of generating and screening allpossible permutations and combinations of antibody structure in aneffort to find the antibody with the maximum efficiency. Conversely,complete randomization of a single 10 amino acid residue CDR wouldgenerate over 10 trillion variants, a number virtually impossible toscreen.

This iterative method can be used to generate double and triple aminoacid replacements in a stepwise process so as to narrow the search forantibodies having higher affinity.

Conversely, it should be recognized that not all locations within thesequences of the different antibody domains may be equal. Substitutionsof any kind in a particular location may be helpful or detrimental. Inaddition, substitutions of certain kinds of amino acids at certainlocations may likewise be a plus or a minus as it affects affinity forthe particular antigen. For example, it may not be necessary to try allpossible hydrophobic amino acids at a given position. It may be that anyhydrophobic amino acid will do as well. Alternatively, an acidic orbasic amino acid at a given location may provide large swings inmeasured affinity. It is therefore necessary also to learn the “rules”of making such substitutions but the determination of such “rules” maynot require the study of all possible combinations andsubstitutions—trends may become apparent after examining fewer than themaximum number of substitutions.

In accordance with the foregoing, the antibodies of the presentinvention are ultra high affinity neutralizing antibodies, such asanti-RSV antibodies, and in the latter case most preferably antibodieswith specificity toward the same epitope of RSV as the antibody of U.S.Pat. No. 5,824,307.

In addition, the affinities of the ultra high affinity antibodies of theinvention typically are at least 10¹⁰ M⁻¹. Because such high affinitiesare not easily measured, except by the procedures described herein, suchvalue may commonly be considered as part of a range and may, forexample, be within 2 fold of 10¹⁰ M⁻¹ or be greater than 10¹⁰ M⁻¹ or mayeven be numerically equal to 10¹⁰ M⁻¹. In such cases, the affinity isdenoted by an affinity constant, which is in the nature of a bindingconstant so as to give units of reciprocal molarity. As such, theaffinity of the antibody for antigen is proportional to the value ofthis constant (i.e., the higher the constant, the greater the affinity).Such a constant is readily calculated from the rate constants for theassociation-dissociation reactions as measured by standard kineticmethodology for antibody reactions.

In a specific embodiment, the high affinity neutralizing antibodies ofthe present invention, and active fragments thereof, have affinityconstants for their respective antigens of at least at least 10¹¹ M⁻¹,in some cases being in excess of this value, a range at the very upperregion of measurability.

The high affinity neutralizing antibodies of the present invention, andactive fragments thereof, may be advantageously directed to anyantigenic determinants desired, such as epitopes present on any type ofmacromolecule, especially peptide epitopes present as part of the threedimensional structure of protein and polypeptide molecules. Such peptideepitopes may commonly be present on the surfaces, or otherwise be partof the structure of, microorganisms and cells, such as cancer cells.Microorganisms expressing such peptide epitopes includes bacteria andviruses. In the latter case, the proteins and polypeptides exhibitingpeptide epitopes may be molecules expressed on the surfaces of the virusor may be expressed by cells infected with a virus. For purposes ofevaluating the efficacy of the rules and methods disclosed according tothe present invention, a virus was chosen as one available antigenicsource for developing antibodies within the present invention. The viruschosen for further analysis was the respiratory syncytial virus (RSV).This latter virus was chosen because it is well characterized withrespect to its replicative cycle as well as with respect to theantigenic determinants found on its surface. In addition, antigens knownto be expressed by cells infected with this virus are wellcharacterized. Thus, the virus exhibits both a surface G antigen and asurface F antigen, both proteins. The G antigen facilitates binding ofthe virus to cell surfaces and the F proteins facilitates the fusion ofthe virus with cells. The cells so infected also express the F antigenon their surfaces and the latter result induces fusion of the cells toform a syncytium, hence the name of the virus. In addition, this virusis a convenient subject for analysis in that cell lines readily infectedby this virus are well known and well characterized, thereby making itan easy virus to grow and culture in vitro. Further, antibodiesavailable for treatment of this virus are known and are commerciallyavailable (for example, the antibodies disclosed in U.S. Pat. No.5,824,307). For these reasons, the antibody disclosed in said patent,the disclosure of which patent is hereby incorporated by reference inits entirety, contains the amino acid sequences of the “referenceantibody” disclosed in FIG. 1 and whose CDR sequences are summarized inTable 1. Thus, the availability of a commercially successful antibodyproduct as well as the well characterized properties of RSV made this anideal combination to use in testing and optimizing the antibodies of thepresent invention and the rules disclosed herein for producing highaffinity CDRs for use in constructing such antibodies. Otherwise, theantibodies disclosed herein, as well as the methods disclosed herein forpreparing such antibodies, are in no way limited to RSV as the antigenbut are readily and advantageously applied generally to the field ofantibody technology. In addition, even the specific embodiments providedby the present disclosure and which are directed specifically toantigenic determinants expressed by RSV, and cells infected with RSV,also may have high affinity for other epitopes, especially those presenton related viruses. Therefore, as disclosed in accordance with thepresent invention, RSV, and the antibody with variable sequences asshown in FIG. 1, are merely a convenient model system used as areference point for developing and applying the methods of antibodytechnology taught by the present invention.

A high affinity neutralizing antibody according to the presentinvention, including active fragments thereof, comprises at least onehigh affinity complementarity determining region (CDR) wherein said CDRhas an amino acid sequence selected to result in an antibody having anaffinity constant (K_(a)) of at least 10¹⁰M⁻¹. In preferred embodiments,such antibody, or active fragment, comprises at least 2 high affinityCDRs, or at least 3 high affinity CDRs or even at least 4 high affinityCDRs. In highly preferred embodiments, such antibodies or activefragments comprise 3 or 4 high affinity CDRs. In one preferredembodiment, such active fragment is an Fab fragment.

The high affinity antibodies of the present invention commonly comprisea mammalian, preferably a human, constant region and a variable region,said variable region comprising heavy and light chain framework regionsand heavy and light chain CDRs, wherein the heavy and light chainframework regions are derived from a mammalian antibody, preferably ahuman antibody, and wherein the CDRs are derived from an antibody ofsome species other than a human, preferably a mouse. Where the frameworkamino acids are also derived from a non-human, the latter is preferablya mouse.

In addition, high affinity antibodies of the invention bind the sameepitope as the antibody from which the CDRs are derived, and wherein atleast one of the CDRs of said ultra high affinity antibody containsamino acid substitutions, and wherein said substitutions comprise thereplacement of one or more amino acids in the CDR regions bynon-identical amino acids, preferably the amino acids of thecorrespondingly aligned positions of the CDR regions of the humanantibody contributing the framework and constant domains.

The high affinity CDRs may be produced by amino acid substitutions innon-high affinity CDRs to produce such high affinity CDRs or such highaffinity CDRs may be synthesized directly to form such high affinityCDRs. Thus, the ultra high affinity neutralizing antibodies of thepresent invention may have amino acid substitutions in only one of theCDR regions, preferably more than one CDR region, and most preferably 3or even 4 such regions, with possibly as many as 5 or even all 6 of theCDRs containing at least one substituted amino acid.

In applying the methods disclosed herein to produce antibodies of thepresent invention, the method of preparing the antibodies is not alimiting factor. Thus, the high affinity neutralizing antibodies of thepresent invention may be prepared by generating polynucleotide sequencescoding for the polypeptides of the antibodies and using vectors toinsert said polynucleotide sequences into permissive cells capable ofnot only expressing such polypeptides but also of assembling them intocharacteristic tetrameric antibody structures that are then retrievedfrom the cells or cell cultures, possibly being secreted into the mediumby such cells. Technologies for such manufacturing procedures arealready known and patented and are not essential to practicing thepresent invention. [see: Morrison et al, U.S. Pat. No. 5,807,715]. Inaddition, the polypeptide chains of the antibodies of the presentinvention may be synthesized chemically, with or without the addition ofenzymes, and then chemically joined to form tetrameric structures of theusual H₂L₂ configuration. Thus, any method of preparing the antibodiesdisclosed herein can be utilized.

The present invention is also directed to the formation of high affinityneutralizing antibodies, with the properties already enumerated, havinghigh affinity as a result predominantly of having high affinity CDRsequences. In accordance with the present invention, the CDR sequencesof the antibodies disclosed herein have been optimized so as to conferupon the antibody molecule the ultra high affinities characteristic ofthe antibodies of the present invention. Such CDR sequences, togetherwith the framework sequences disclosed herein, especially those taughtby the sequences of FIGS. 1, 3, 4, 5, 6, and 7, and most especially whenused with constant region sequences characteristic of the antibodies ofthe organism acting as recipient of the antibodies of the presentinvention when used therapeutically, produce the antibodies of thepresent invention in their more specific embodiments. However, themethods of the present invention are more specifically directed to theCDR amino acid sequences.

To produce immunoglobulins, such as antibodies and/or active fragmentsthereof, within the present invention, e.g., high affinity neutralizingantibodies, the rules taught by the present invention are usedadvantageously to produce antibody molecules whose structuresincorporate the sequences of the high affinity CDR sequences disclosedaccording to the present invention. Thus, the high affinity neutralizingantibodies of the present invention are, in essence, not truly“monoclonal” antibodies as that term is commonly used, since they do nothave to be produced by cloning anything. As already mentioned, thesequences of the antibodies of the invention may be directly synthesizedand thus may not be identical to any antibody sequences, especially notto any CDR sequences, presently known. The sequences themselves can bewholly novel ab initio and not be exactly represented in any antibodyproduced by any living organism. Thus, the high affinity CDR sequencesdisclosed herein are found, or achieved, by optimization, as disclosedherein, and then, once said high affinity CDR sequences are known, canbe used to synthesize fully functional antibody molecules, whetherdimeric or tetrameric, bifunctional or monofunctional, by any and allmeans known to science.

In keeping with the foregoing, and in order to better describe thesequences disclosed according to the invention, including theiroptimization, the sequence of the light and heavy chain variable regionsof a reference antibody (here, the anti-RSV antibody of U.S. Pat. No.5,824,307) are shown in FIG. 1A (light chain variable region—SEQ IDNO: 1) and FIG. 1B (heavy chain variable region—SEQ ID NO: 2). Also inaccordance with the invention, novel sequences were produced with aminoacid differences only in CDR regions relative to the reference antibody.One means utilized to accomplish this result was to introduce mutationsin CDR regions of the so-called starting or reference chains and thenassay the resulting recombinatorial clones for antigen (RSV F protein)affinity.

In accordance with the forgoing, changes were made in first one CDRsequence to optimize that sequence and determine the “critical” residue,or residues, than said position(s) was optimized through a series ofamino acid substitutions limited to that position alone. Each of the 6CDRs of the antibody clone was studied in turn until the “critical” CDRswere determined (wherein “critical CDRs” means CDRs having a substantialeffect on antibody binding, such as the beneficial or high affinity CDRsof Table 3). No all CDRs were found to be critical. For the antibodyused in this particular study, only CDRs H1, H3, L2, and L3 were foundto be critical but the results may be different for a differentantibody. Once a “high affinity” CDR was determined (i.e., a “beneficialCDR”) then combinations of the CDRs were studied to optimize thecombination of CDR sequences resulting in a high affinity neutralizingantibody of the invention.

As a very specific embodiment, the invention disclosed herein relates toa high affinity neutralizing antibody against respiratory syncytialvirus (RSV) having an affinity constant of at least 10¹⁰ M⁻¹, whereinsaid affinity constant could be within at least 2 fold of this valuebecause of the variability of such determinations and the variability ofaffinity of the different cloned antibodies for the antigen (here, Fantigen of RSV). Some of the resulting optimized structures within thisembodiment had K_(a) greater than 10¹¹ M⁻¹ (for example, clones numbered22 and 23 in Table 4).

This high affinity neutralizing antibody is also an antibody that bindsto the same epitope on RSV as the antibody whose light chain variableregion has the sequence of SEQ ID NO: 1 (FIG. 1A) and whose heavy chainvariable region has the sequence of SEQ ID NO: 2 (FIG. 1B).

In general, the approach used to identify antibodies of the invention,based on the specific example of anti-RSV just described, was togenerate nucleotide sequences for the genes expressing the desiredantibody chains and insert these into vectors then used to transformEscherichia coli cells by standard protocols. The cells were grown inwells and the supernatant sampled and measured for antigen binding usingcapture lift and ELISA techniques. [See: Watkins et al, (1997) Anal.Biochem. 253, 37-45; Watkins et al, (1998) Anal. Biochem. 256, 169-177(the specifications of which are incorporated herein by reference)]These polynucleotides were designed so as to provide single amino acidreplacements in the CDRs that could then be screened for increasedaffinity, with beneficial replacements (those yielding increasedaffinity) being selectively combined for increased affinity. These werethen screened for binding affinity for F antigen of RSV versus the basicor reference antibody.

Using this protocol, ELISA data indicated that no single amino acidreplacements in CDRs L1 or H2 produced any increase in the affinity ofthe antibody clone for the epitope used as antigen (here, the F antigenof RSV). Therefore, the antibodies of the present invention all containCDR sequences that differ from the reference antibody only in CDRs L2,L3, H1 and H3 (here, the reference antibody with sequences in FIG. 1 wasmerely a useful reference against which to test procedures foroptimization of antibody affinity by increasing K_(a) and any othersystem could be used equally well).

The antibodies thus disclosed herein with respect to RSV also commonlyhave framework regions derived from a human antibody but, where not soderived, preferably from a mouse.

For the CDRs of the reference antibody, the amino acid sequence of eachCDR (as given in the sequences of FIG. 1) is shown in Table 1. Aminoacid residue locations within the CDRs of the basic or referenceantibody, which, if replaced by amino acids as taught by the presentinvention, following optimization, produced high affinity CDR sequences(resulting in very high affinity neutralizing antibodies) and thereby abeneficial result (increase in affinity) are indicated in bold face andunderlining in Table 1 (such sequences giving increased affinity overthe reference antibody being denoted as “beneficial CDRs” or “highaffinity CDRs”). The CDRs of the basic or reference antibody (FIG. 1)are referred to herein as “basic or reference CDRs”). Thus, Table 1represents the CDR sequences depicted in FIG. 1 (i.e., for the anti-RSVreference antibody used herein to monitor optimization).

TABLE 1 Sequences of Basic or Reference CDRs SEQ ID CDR Length SequenceNO. L1 10 S A S S S V G Y M H 3 L2 7 D T  S K L A S 4 L3 9 F Q G S  GY P F T 5 H1 7 T  S G M S V G 6 H2 16 D I W W D D K K D Y N P S 7 L K SH3 10 S M I T N  W Y F D V 8

With respect to the sequences disclosed herein, the CDR regions asdefined for purposes of the present invention are those segmentscorresponding to residues 24-33 (CDR L1), 49-55 (CDR L2) and 88-95 (CDRL3) of the light chain variable regions and residues 31-37 (CDR H1),52-67 (CDR H2) and 100-109 (CDR H3) of the heavy chain variable regionsof the antibodies disclosed herein.

In producing the antibodies of the present invention, whether bygenerating clones or cloning the polypeptide chains composing saidantibodies, or by direct synthesis of the polypeptide sequences, with orwithout the use of polynucleotide sequences coding therefor, or bywhatever method the user may choose, since no method of producing saidantibodies results in a limitation of the teaching of the presentinvention, the basic or reference antibody (heavy and light chainvariable regions (CDRs plus Framework) shown in FIG. 1) can be used as a“template” for generating the novel CDR sequences of the antibodies ofthe present invention and for purposes of comparing binding constants,etc. Standard approaches to characterizing and synthesizing the six CDRlibraries of single mutations were used (see Wu et al, Proc. Natl. Acad.Sci. 95:6037-6042 (1998)). The target CDR was first deleted for each ofthe libraries prior to annealing the nucleotides. For synthesis of thelibraries, the CDRs were defined as in Table 1. Codon based mutagenesisfor oligonucleotide synthesis to yield the CDR sequences of theinvention was employed (as described above).

Libraries were initially screened by capture lift to identify thehighest affinity variants. Subsequently, these clones were furthercharacterized using capture ELISA and by titration on immobilizedantigen.

DNA from the highest affinity variants was sequenced to determine thenature of the beneficial or high affinity replacements. After screening,it was determined that eight beneficial or high affinity replacements,occurring in only four of the CDRs, had been observed. These aresummarized as the CDR sequences in Table 3 with differences versus thereference or basic CDRs of Table 1 being bold and underlined. Thus, theCDR sequences of Table 3 can be considered a CDR library of cassettesavailable for use in producing a high affinity neutralizing antibody ofthe present invention where specificity is directed toward the F antigenof RSV.

Analysis of the data indicated that replacement of amino acids atselected locations had greatly increased epitope binding, especiallywhere the nature of the replacement was the insertion of an amino acidselected from the group phenylalanine, alanine, proline, tryptophan andtyrosine, most especially phenylalanine, all such beneficial or highaffinity replacements again being at selected positions.

For the optimization experiment described herein and employing theRSV/anti-RSV system, the most beneficial of high affinity CDRs werefound to result from amino acid replacements in 3 or 4 of the 6 CDRs,and in just 4 amino acid locations overall. Thus, the high affinityneutralizing antibodies of the present invention contain amino acidsequences differing from that of the base or reference antibody only incomplementarity determining regions, or in such regions as well as insurrounding framework regions, unlike previously known man-madeantibodies. Thus, the antibodies of the present invention are highaffinity neutralizing antibodies containing one or more CDR sequencesselected so as to produce high affinity in the antibody molecule. In thespecific embodiment using RSV as the target such differences are foundonly in L2 (or CDRL2), L3 (or CDRL3), H1 (or CDRH1) and H3 (or CDRH3).As noted, the greatest affinities for this antibody occurred only atselected amino acid positions of these CDRs, and in some of theembodiments of the present invention just one amino acid location ineach CDR was preferred for giving high affinity.

Thus, for CDR H1, substitution at amino acid 2 of the CDR (counting fromthe amino terminal end of the particular underlined CDR sequence of FIG.1B), especially by replacing the serine located at position 2 of CDR H1of the basic or reference antibody with either an alanine or a proline,was found to be most beneficial and therefore to result in higheraffinity for the RSV antigen epitope. For CDR H3, replacement of theglycine at position 6 of the CDR sequence, especially by either aphenylalanine or tryptophan, most especially by phenylalanine, was foundto result in increased affinity for the RSV epitope. For CDR L2,replacement of the serine at position 3 of the CDR, especially by eithera phenylalanine or a tyrosine, resulted in increased affinity for Fantigen. For CDR L3, replacement of the glycine at position 5 of theCDR, especially by phenylalanine, tryptophan or tyrosine, resulted inincreased affinity for the RSV epitope.

In accordance with the invention, by combining such amino acidsubstitutions so that more than one occurred in the same antibodymolecule, it was possible to greatly increase the affinity of theantibodies disclosed herein for the epitope of the F antigen of RSV.

Table 2 shows the results of using the novel CDR sequences (for H1, H3,L2, and L3, respectively) of a number of clones according to the presentinvention. As shown in Table 2, a particular antibody may haveincorporated therein as many as 1, 2, or 3 novel CDRs of the inventionversus the basic or reference antibody chains shown in FIGS. 1A and 1B(for light (L2 and L3) and heavy (H1 and H3) chains, respectively). Theeffects of the novel CDRs are described in terms of Antigen (Ag)titration score (see below), where the basic or reference antibody isshown at the top and has a score of 0.1. Other scores are indicatedrelative to this 0.1 score of the “basic or reference antibody”sequences. The identities of the amino acids giving highest affinitiesat the respective locations (i.e., position 2 for H1, position 6 for H3,position 3 for L2, and position 5 for L3) are indicated below the aminoacids for the basic or reference antibody merely to indicate the rangeof mutations used for each CDR.

The total number of clones examined in this experiment was 37, with someduplicates (indicated by the “n” value in parenthesis, for example,“n=4” for clone No. 7 indicates that 4 duplicate clones were examined).

In general, the data showed that there is a correlation between affinityand the number of beneficial or high affinity CDRs, with all of thehigher affinity variants having more than one beneficial or highaffinity CDR. Further, all of the best clones had an F (Phe) at position6 within CDR H3. Also, the beneficial or high affinity CDRs were thosewherein a hydrophobic, especially an aromatic, amino acid was insertedin place of the residue found in the basic or reference antibody.

The antibody titration assay employed varying concentrations of antibodyusing 500 ng of RSV F antigen for each measurement. A graph ofcomparison data for a number of the combinatorial clones of Table 2 isshown in FIG. 2.

In sum, Table 2 shows a number of clones evaluated by the proceduresdescribed herein along with the amino acids occurring at the keylocations (underlined and bold-faced in Tables 1 and 3) of CDRs H1, H3,L2, and L3. The Table also summarizes the number of differences betweenthe CDRs of these clones versus the corresponding CDRs of the referenceantibody (See Table 1 and FIG. 1). The right side of the Table shows an“Ag Score” or antigen binding value, which represents an arbitrary andqualitative value, ranging from 0-4, and represents a qualitativeestimate of the relative binding ability of the different antibodyclones based on their respective titration curves. This value isprovided here only for rough qualitative comparisons of the differentantibodies and is not intended as a quantitative measure of bindingability.

TABLE 2 Summary of Clone Data CDRs Clone H1 H3 L2 L3 # Novel CDRs AgScore Basic S W S G 0 0.1 Single A F F F P Y W Y  1 A F S F 3 4  2 A F FG 3 4  3 (n = 3) P F F F 4 4  4 (n = 3) P F F Y 4 3.5  5 (n = 3) P F F W4 3.5  6 P F Y F 4 3.5  7 (n = 4) P F F G 3 3  8 P F F ? 3+ 3.5  9 (n =2) P F S W 3 3 10 P F S F 3 3 11 P W F W 3 3 12 (n = 2) P W F F 3 2.5 13(n = 3) S F F F 3 2.5 14 S F F W 3 2.5 15 (n = 2) A F S G 2 2.5 16 (n =2) P F S G 2 2 17 P W S W 2 2 18 (n = 2) S F F G 2 2 19 S F S W 2 2 20 SF S F 2 2 21 S W Y F 2 2

Table 2 also shows the number of novel CDRs for each antibody clone(meaning the number of CDRs in the antibody with at least one amino aciddifference with respect to the corresponding CDR of the referenceantibody—see Table 1). The number of novel CDRs is also the number of“beneficial” or “high affinity” CDRs present in that antibody molecule.The amino acid differences in the novel CDR would occur at the positionbold and underlined in Table 1 for the reference antibody so that theamino acid bold and underlined in Table 1 has been replaced by the aminoacid indicated for the respective CDR in Table 2 (using standard singleletter amino acid designations).

The novel CDRs represented in each of the clones is readily determinedby locating the clone in the table, and matching the indicated aminoacid for each CDR with the corresponding amino acid for the same CDRnext to the basic or reference clone. For convenience, where an aminoacid is different in a particular CDR of one of the clones, the newamino acid is indicated in bold face. In addition, for all clones shownin the table, replacements occur only at the selected locations recitedabove as yielding a novel CDR. Thus, all substitutions in CDR H1relative to the basic or reference antibody are at position 2 of the CDR(meaning, again, the second amino acid from the N-terminal end of CDR H1as underlined and bold-faced in FIG. 1 and Tables 1 and 3), allsubstitutions in CDR H3 are at position 6, all substitutions in CDR L2are at position 3, and all substitutions in CDR L3 are at positions 5,again all with reference to the basic or reference antibody. It shouldbe kept in mind that the basic or reference antibody was chosen becauseit was known already to have a very high affinity for RSV-epitopes.[See: Johnson et al, (1997) J. Infect. Dis., 176, 1215-1224] So, forexample, the table shows that for clone No. 1, for the beneficial orhigh affinity CDR H1, an alanine is used in place of the serine atposition 2 of CDR H1 of the basic or reference antibody, therebyachieving an increased affinity for RSV, and a phenylalanine occurs inplace of the tryptophan at position 6 of CDR H3, the serine at position3 of CDR L2 of the basic or reference antibody was used and aphenylalanine occurred at position 5 of beneficial or high affinity CDRL3.

Thus, the novel and beneficial CDRs according to the present invention(i.e., high affinity CDRs or CDR sequences whose presence in the basicor reference antibody in place of the corresponding basic or referenceCDR served to greatly increase the affinity of said antibody for thesame RSV epitope) which are present in the antibody structures producedin the supernatants tested for the clones of Table 2 are summarized inTable 3. In each case, the bold face indicates how the novel andbeneficial, or high affinity, CDRs of the invention differ from thecorresponding CDRs of the basic or reference anti-RSV antibody.

TABLE 3 Sequences for High Affinity CDRs CDR Sequence SEQ ID NO: H1 T AGMSVG 9 H1 T P GMSVG 10 H3 SMITN F YFDV 11 L2 DT F KLAS 12 L2 DT Y KLAS13 L3 FQGS F YPFT  14 L3 FQGS Y YPFT  15 L3 FQGS W YPFT  16

While the CDR sequences of Table 3 represent the sequences for the highaffinity CDRs disclosed according to the invention, it is understoodthat one or more of these CDRs may be present in the same antibody andthe sequences of the table indicate the set from which appropriatesequences for each of the high affinity CDRs may be selected. Thus, asshown in Table 3, when a high affinity H1 CDR is present in a highaffinity neutralizing antibody of the invention disclosed herein, it hasa sequence corresponding to the sequence of SEQ ID NO: 9 or 10. When aneutralizing antibody of the claimed invention contains a high affinityH3 CDR, said CDR has the sequence of SEQ ID NO: 12. When a high affinityneutralizing antibody of the invention contains a high affinity L2 CDR,said high affinity L2 CDR has an amino acid sequence selected from thegroup consisting of the sequences of SEQ ID NO: 12 and 13. Finally, whena high affinity neutralizing antibody of the present invention containsa high affinity L3 CDR, said CDR has an amino acid sequence selectedfrom the group consisting of the sequences of SEQ ID NO: 14, 15 and 16.

As already stated, in one embodiment, the high affinity neutralizingantibodies are antibodies that include a human constant region.

Thus, in a preferred embodiment, the high affinity neutralizing antibodyof the invention, with an affinity of at least 10¹⁰ M⁻¹, or even atleast 10¹¹ M⁻¹, is a grafted antibody that includes a human constantregion and a framework for the heavy and light chains wherein at least aportion of the framework is derived from a human antibody (or from aconsensus sequence of a human antibody framework).

In another embodiment, all of the framework is derived from a humanantibody (or a human consensus sequence).

Thus, an RSV-neutralizing antibody, with an affinity of at least 10¹⁰M⁻¹, is a grafted antibody having a human constant region, one or moreCDRs that are derived from a non-human antibody in which at least one ofthe amino acids in at least one of said CDRs is changed and in which allor a portion of the framework is derived from a human antibody (or aconsensus sequence of a human antibody framework).

Because the combination of CDR sequences of one antibody with non-CDRregions of another antibody results from a form of “grafting” of CDRsonto the remainder of the molecule, these have been referred to as “CDRgrafted” antibodies. Today, using the techniques of genetic engineeringthe same product can be formed without isolating any sequences fromactual antibodies. So long as the desired CDR sequences, and theconstant and framework sequence are known, genes with the desiredsequences can be assembled and, using a variety of vectors, insertedinto appropriate cells for expression of the functional tetramericantibody molecules. Coupling this with the methodology already describedpermits the assembly of single mutation libraries wherein the antibodiespossess the same sequences as corresponding grafted antibodies and,therefore, the same structure and binding affinities.

The high affinity antibodies of the invention can be present in arelatively pure or isolated form as well as in a supernatant drawn fromcells grown in wells or on plates. Such supernatants were used togenerate the data of Table 1. The antibodies of the invention can thusalso be present in the form of a composition comprising the antibody ofthe invention and wherein said antibody is suspended in apharmacologically acceptable carrier, or excipient. The antibodies ofthe invention may be present in such a composition at a concentration,or in an amount, sufficient to be of therapeutic or pharmacologicalvalue in treating diseases, such as RSV. Said antibodies may also bepresent in a composition in a more dilute form.

Consequently, the invention is also directed to providing a method ofpreventing and/or treating respiratory syncytial virus infectionscomprising the administering to a patient at risk thereof, or afflictedtherewith, of a therapeutically (including prophylactically) effectiveamount of the antibody composition described herein.

One preferred embodiment of the high affinity antibodies of the presentinvention is the antibody whose heavy and light chain CDR regions havesequences as follows: CDR H1 has the sequence of SEQ ID NO: 9, CDR H3has the sequence of SEQ ID NO: 11, CDR L2 has the sequence of SEQ ID NO:4 (no change from CDR L2 of the reference sequence of FIG. 1A), and CDRL3 has the sequence of SEQ ID NO: 14 (see Table 3). In this preferredembodiment, the affinity constant is about 6.99×10¹⁰ (or about 14.3 pMas a dissociation constant) as shown in Table 4 (clone 1). The heavy andlight chain variable regions of an antibody comprising this embodiment,along with framework sequences, is shown in FIG. 3.

Another preferred embodiment of the high affinity antibodies of thepresent invention is the antibody whose heavy and light chain CDRregions have the sequences as follows: CDR H1 has the sequence of SEQ IDNO: 9, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has the sequenceof SEQ ID NO: 12, and CDR L3 has the sequence of SEQ ID NO: 5 (see Table2, clone 2—no difference from the reference sequence of CDR L3 of FIG.1A). In this preferred embodiment, the affinity constant is about7.30×10¹⁰ (or about 13.7 pM as a dissociation constant) as shown inTable 4 (clone 2). The heavy and light chain variable regions of anantibody comprising this embodiment, along with framework sequences, isshown in FIG. 4.

An additional preferred embodiment of the high affinity antibodies ofthe present invention is the antibody whose heavy and light chain CDRregions have the sequences as follows: CDR H1 has the sequence of SEQ IDNO: 10, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has thesequence of SEQ ID NO: 12, and CDR L3 has the sequence of SEQ ID NO: 14(see Table 3 for CDR sequences) which clone is designated number 3 inTable 2. In this preferred embodiment, the affinity constant is about8.13×10¹⁰ (or about 12.3 pM as a dissociation constant) as shown inTable 4 (clone 3). The heavy and light chain variable regions of anantibody comprising this embodiment, along with framework sequences, isshown in FIG. 5.

A most preferred embodiment of the high affinity antibodies of thepresent invention is the antibody whose heavy and light chain CDRregions have the sequences as follows: CDR H1 has the sequence of SEQ IDNO: 9, CDR H3 has the sequence of SEQ ID NO: 11, CDR L2 has the sequenceof SEQ ID NO: 12, and CDR L3 has the sequence of SEQ ID NO: 14 (seeTable 3). In this preferred embodiment, the affinity constant is about3.6×10¹¹ (or about 2.8 pM as a dissociation constant) as shown in Table4 (clone 22). The heavy and light chain variable regions of an antibodycomprising this embodiment, along with framework sequences, is shown inFIG. 6.

Another most preferred embodiment of the present invention is theantibody whose heavy and light chain CDR regions include the followingsequences: CDR H1 has the sequence of SEQ ID NO: 9, CDR H3 has thesequence of SEQ ID NO: 11, CDR L2 has the sequence of SEQ ID NO: 12, andCDR L3 has the sequence of SEQ ID NO: 15 (see Table 3). In this latterpreferred embodiment, the affinity constant is about 4×10¹¹ (about 2.5pM as a dissociation constant) as shown in Table 4 (clone 23). The heavyand light chain variable regions of an antibody comprising thisembodiment, along with framework sequences, is shown in FIG. 7.

In particularly preferred embodiments, the antibodies of the presentinvention will have the framework regions of the sequences depicted forthe framework regions shown in FIGS. 1, 3, 4, 5, 6, and 7 (each containsthe same framework regions and differ only in CDR sequences). These mostpreferred embodiments include the neutralizing antibody wherein thelight chain variable region has the amino acid sequence of SEQ ID NO: 17and the heavy chain variable region has the amino acid sequence of SEQID NO: 18; the neutralizing antibody wherein the light chain variableregion has the amino acid sequence of SEQ ID NO: 19 and the heavy chainvariable region has the amino acid sequence of SEQ ID NO: 20; theneutralizing antibody wherein the light chain variable region has theamino acid sequence of SEQ ID NO: 21 and the heavy chain variable regionhas the amino acid sequence of SEQ ID NO: 22; the neutralizing antibodywherein the light chain variable region has the amino acid sequence ofSEQ ID NO: 23 and the heavy chain variable region has the amino acidsequence of SEQ ID NO: 24; the neutralizing antibody wherein the lightchain variable region has the amino acid sequence of SEQ ID NO: 25 andthe heavy chain variable region has the amino acid sequence of SEQ IDNO: 26.

It should be kept in mind that while the high affinity neutralizingantibodies of the present invention can be assembled from CDR regionsand non-CDR regions derived from actual neutralizing antibodies bysplicing amino acid segments together (and antibodies so assembled wouldbe within the invention disclosed herein) the antibodies of the presentinvention are most conveniently prepared by genetically engineeringappropriate gene sequences into vectors that may then be transfectedinto suitable cell lines for eventual expression of the assembledantibody molecules by the engineered cells. In fact, such recombinantprocedures were employed to prepare the antibodies disclosed herein. Inaddition, because the sequences of the chains of the high affinityantibodies are known from the disclosure herein, such antibodies couldalso be assembled by direct synthesis of the appropriate chains and thenallowed to self-assemble into tetrameric (H₂L₂) bivalent antibodystructures.

The method of preparing the high affinity neutralizing antibodies of theinvention involved the creation of a combinatorial library which wasused to prepare clones producing antibodies comprising the beneficialCDRs of the invention that could then be screened for affinity for RSVepitopes (for Example, FIG. 2).

Example 1 Kinetic Analysis of Humanized RSV Mabs by BIAcore™

The kinetics of interaction between high affinity anti-RSV Mabs and theRSV F protein was studied by surface plasmon resonance using a PharmaciaBIAcore™ biosensor. A recombinant baculovirus expressing a C-terminaltruncated F protein provided an abundant source of antigen for kineticstudies. The supernatant, which contained the secreted F protein, wasenriched approximately 20-fold by successive chromatography onconcanavalin A and Q-sepharose columns. The pooled fractions weredialyzed against 10 mM sodium citrate (pH 5.5), and concentrated toapproximately 0.1 mg/ml. In a typical experiment, an aliquot of theF-protein (100 ml) was amine-coupled to the BIAcore sensor chip. Theamount immobilized gave approximately 2000 response units (R_(max)) ofsignal when saturated with the mouse monoclonal antibodies H1129 orH1308F (prepared as in U.S. Pat. No. 5,824,307, whose disclosure ishereby incorporated by reference). This indicated that there was anequal number of “A” and “C” antigenic sites on the F-protein preparationfollowing the coupling procedure. Two unrelated irrelevant Mabs (RVFV4D4 and CMV H758) showed no interaction with the immobilized F protein.A typical kinetic study involved the injection of 35 ml of Mab atvarying concentrations (25-300 nM) in PBS buffer containing 0.05%Tween-20 (PBS/Tween). The flow rate was maintained at 5 ml/min, giving a7 min binding phase. Following the injection of Mab, the flow wasexchanged with PBS/Tween buffer for 30 min for determining the rate ofdissociation. The sensor chip was regenerated between cycles with a 2min pulse of 10 mM HCl. The regeneration step caused a minimal loss ofbinding capacity of the immobilized F-protein (4% loss per cycle). Thissmall decrease did not change the calculated values of the rateconstants for binding and dissociation (also called the k_(on) andk_(off), respectively).

More specifically, for measurement of k_(assoc) (or k_(on)), F proteinwas directly immobilized by the EDC/NHS method(EDC=N-ethyl-N′-[3-diethylamino-propyl]carbodiimide;NHS=N-hydroxysuccinimide) with the F-protein being injected over theEDC/NHS activated sensor chip. Briefly, 4 μg/ml of F protein in 10 mMNaOAc, pH 4.0 was prepared and about a 30 μl injection gives about 500RU (response units) of immobilized F protein under the above referencedconditions. The blank flow cell (VnR immobilized-CM dextran surface) wassubtracted for kinetic analysis. The column could be regenerated using100 mM HCl (with 72 seconds of contact time being required for fullregeneration). This treatment removed bound Fab completely withoutdamaging the immobilized antigen and could be used for over 40regenerations. For k_(on) measurements, Fab concentrations were 12.5 nM,25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. The dissociation phase wasanalyzed from 230 seconds (30 seconds after start of the dissociationphase) to 900 seconds. Kinetics were analyzed by 1:1 Langmuir fitting(global fitting). Measurements were done in HBS-EP buffer (10 mM HEPES,pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20.

For measurements of combinatorial clones, as disclosed herein, thek_(on) and k_(off) were measured separately. The k_(on) was measured atconditions that were the same as those for the single mutation clonesand was analyzed similarly.

For measuring k_(dissoc) (or k_(off)), the following conditions wereemployed. Briefly, 4100 RU of F protein were immobilized (as above) withCM-dextran used as the blank. Here, 3000 RU of Fab was bound (withdissociated Fab high enough to offset machine fluctuation). HBS plus 5nM F protein (about 350-2000 times higher than the K_(dissoc) orK_(d)—the dissociation equilibrium constant) was used as buffer. Thedissociation phase was 6-15 hours at a flow rate of 5 μl/min. Under theconditions used herein, re-binding of the dissociated Fab was minimal.For further details, see the manual with the biosensor.

The binding of the high affinity anti-RSV antibodies to the F protein,or other epitopic sites on RSV, disclosed herein was calculated from theratio of the first order rate constant for dissociation to the secondorder rate constant for binding or association(K_(d)=k_(diss)/k_(assoc)). The value for k_(assoc) was calculated basedon the following rate equation:

dR/dt=k _(assoc) [Mab]R _(max)−(k _(assoc) [Mab]+k _(diss))R

where R and R_(max) are the response units at time t and infinity,respectively. A plot of dr/dt as a function of R gives a slope of(k_(assoc)[Mab]+k_(diss))—Since these slopes are linearly related to the[Mab], the value k_(assoc) can be derived from a replot of the slopesversus [Mab]. The slope of the new line is equal to k_(assoc). Althoughthe value of k_(diss) can be extrapolated from the Y-intercept, a moreaccurate value was determined by direct measurement of k_(diss).Following the injection phase of the Mab, PBS/Tween buffer flows acrossthe sensor chip. From this point, [Mab]=0. The above stated equation fordR/dt thus reduces to:

dr/dt=k or dR/R=k _(diss) dt

Integration of this equation then gives:

In(R ₀ /R _(t))=k _(diss) t

where R₀/R_(t)) are the response units at time 0 (start of dissociationphase) and t, respectively. Lastly, plotting In(R₀/R_(t)) as a functionof t gives a slope of k_(diss).

In the preferred embodiment herein, the numerical values from suchantibody variants were as follows:

TABLE 4 Summary of Kinetic Constants from Ultra-high AffinityAntibodies. Clone ID CDRs k_(assoc) (M⁻¹sec⁻¹) k_(diss) (sec⁻¹) K_(a)(M⁻¹) 1 AFSF 1.13 × 10⁵ 1.62 × 10⁻⁶ 6.98 × 10¹⁰ 2 AFFG 1.33 × 10⁵ 1.82 ×10⁻⁶ 7.31 × 10¹⁰ 3 PFFF 1.10 × 10⁵ 1.35 × 10⁻⁶ 8.15 × 10¹⁰ 22 AFFF 1.34× 10⁵ 3.70 × 10⁻⁷ 3.62 × 10¹¹ 23 AFFY 1.22 × 10⁵ 3.03 × 10⁻⁷ 4.03 × 10¹¹

Here, the CDRs represent the amino acids replacing the reference aminoacids at the key positions (or critical positions) of the CDRs shown inTable 1 (in bold and underlined) for a reference antibody. Thus, forexample, clone 22 has an alanine at position 2 of CDR H1 (residue 32 ofthe heavy chain variable region—SEQ ID NO: 24) in place of the serineshown at that position in Table 1 (SEQ ID NO: 6), a phenylalanine atposition 6 of CDR H3 (residue 105 of the heavy chain variable region—SEQID NO: 24) in place of the tryptophan shown at that position in Table 1(SEQ ID NO: 8), a phenylalanine at position 3 of CDR L2 (residue 51 ofthe light chain variable region—SEQ ID NO: 23) in place of the serineshown at that position in Table 1 (SEQ ID NO: 4), and a phenylalanine atposition 5 of CDR L3 (residue 92 of the light chain variable region—SEQID NO: 23) in place of the glycine shown at that position in Table 1(SEQ ID NO: 5).

Of course, in forming such clones, Table 3 represents a pool ofpotential CDRs from which the high affinity CDRs of the antibodies ofthe present invention are to be drawn. For example, clone 23 of Table 4uses the same CDRs as clone 22 with the exception of CDR L3, which has atyrosine at position 5 of CDR L3 (Table 3—SEQ ID NO: 15) in place of theglycine shown in that position in Table 1 (SEQ ID NO: 5). Thesubstitutions at the corresponding critical positions are likewise shownin Table 2.

Example 2 Microneutralization Assay

Neutralization of the antibodies of the present invention weredetermined by microneutralization assay. This microneutralization assayis a modification of the procedures described by Anderson et al (1985).Antibody dilutions were made in triplicate using a 96-well plate. TenTCID₅₀ of respiratory syncytial virus (RSV—Long strain) were incubatedwith serial dilutions of the antibody (or Fabs) to be tested for 2 hoursat 37° C. in the wells of a 96-well plate. RSV susceptible HEp-2 cells(2.5×10⁴) were then added to each well and cultured for 5 days at 37° C.in 5% CO₂. After 5 days, the medium was aspirated and cells were washedand fixed to the plates with 80% methanol and 20% PBS. RSV replicationwas then determined by F protein expression. Fixed cells were incubatedwith a biotin-conjugated anti-F protein monoclonal antibody (pan Fprotein, C-site-specific MAb 133-1H) washed and horseradish peroxidaseconjugated avidin was added to the wells. The wells were washed againand turnover of substrate TMB (thionitrobenzoic acid) was measured at450 nm. The neutralizing titer was expressed as the antibodyconcentration that caused at least 50% reduction in absorbency at 450 nm(the OD₄₅₀) from virus-only control cells. Results for severalantibodies of the present invention are shown by the graph in FIG. 8while results using Fab fragments are depicted in the graph of FIG. 9.

BACKGROUND AND CITED REFERENCES

-   1. Hall, C. B., Douglas, R. G., Geiman, J. M. et al., N. Engl. J.    Med. 293:1343, 1975.-   2. Hall, C. B., McBride, J. T., Walsh, E. E. et al., N. Engl. J.    Med. 308:1443, 1983.-   3. Hall, C. B., McBride, J. T., Gala, C. L. et al., JAMA 254:3047,    1985.-   4. Wald, E. R., et al., J. Pediat. 112:154, 1988.-   5. Kapikian, A. Z., Mithcell, R. H., Chanock, R. M. et al., Am. J.    Epidemiol. 89:405, 1969.-   6. Prince, G. A., Hemming, V. G., Horswood, R. L. et al., Virus Res.    3:193, 1985.-   7. Hemming, V. G., Prince, G. A., Horswood, R. L. et al., J. Infect.    Dis. 152:1083, 1985.-   8. Wright, P. F., Belshe, R. B., et al., Infect. Immun. 37:397,    1982.-   9. Conrad, D. A., Christenson, J. C., et al., Peditr. Infect.    Dis. J. 6:152, 1987.-   10. LoBuglio, A. F., Wheeler, R. L., Trang, J. et al., Proc. Natl.    Acad. Sci. 86:4220, 1989.-   11. Steplewski, Z., Sun, L. K., Shearman, C. W. et al., Proc. Natl.    Acad. Sci. 85:4852, 1988.-   12. Boulianne, G. L, Hozumi, N., Shulman, M. J. Nature. 312:643,    1984.-   13. Sun, L. K., Curtis, P., Rakowicz-Szulczynska, E. et al., Proc.    Natl. Acad. Sci. 84:214, 1987.-   14. Liu, A. Y., Mack, P. W., Champion, C. I., Robinson, R. R., Gene    54:33, 1987.-   15. Morrison, S. L., Johnson, M. J., Hersenber, L. A., Oi, V. T.    Proc. Natl. Acad. Sci. 81:6851, 1984.-   16. Morrison, S. L. Science 229:1202, 1985.-   17. Sahagan, B. G., Dorai, H., Saltzgaber-Muller, J. et al, J.    Immunol. 137:1066, 1986.-   18. Taked, S., Naito, T., Hama, K., Noma, T., Honjo, T., Nature    314:452, 1985.-   19. Carson, D. A., Freimark, B. D., Adv. Immunol. 38:275, 1986.-   20. Beeler, J. A., et al., J. Virol. 63:2941-2950, 1989.-   21. Coelingh, et al., Virology, 143:569-582, 1985.-   22. Anderson et al. Microneutralization test for respiratory    syncytial virus based on an enzyme immunoassay, J. Clin.    Microbiol. (1985) 22:1050-1052.-   23. Johnson et al., Development of a humanized monoclonal antibody    (MEDI-493) with potent in vitro and in vivo activity against    respiratory syncytial virus, J. Infectious Diseases (1997) 176:    1215-1224.

1.-48. (canceled)
 49. An isolated high affinity neutralizingimmunoglobulin that specifically binds to a respiratory syncytial virus(RSV) F antigen with an affinity constant (K_(a)) of at least 10¹⁰ M⁻¹as measured by surface plasmon resonance, wherein the high affinityneutralizing immunoglobulin binds to the same epitope on the RSV Fantigen as an antibody comprising a heavy chain variable region (VH)having an amino acid sequence SEQ ID NO:2 (FIG. 1B) and a light chainvariable region (VL) having the amino acid sequence SEQ ID NO:1 (FIG.1A).
 50. An isolated high affinity neutralizing immunoglobulin thatspecifically binds to a RSV F antigen with an affinity constant (K_(a))of at least 10¹⁰ M⁻¹ as measured by surface plasmon resonance, whereinthe immunoglobulin comprises one or more amino acid changes in one ormore complementarity determining regions (CDRs) as compared to anexisting antibody, wherein the existing antibody comprises: a. a VLcomprising the following CDR sequences:

b. a VH comprising the following CDR sequences:

and wherein one or more amino acid residue substitutions are made at theboxed positions, such that the amino acid substitutions have the effectof producing an increase in the K_(a) of the antibody.
 51. The highaffinity neutralizing immunoglobulin of claim 50, wherein the highaffinity neutralizing immunoglobulin binds to the same epitope on theRSV F antigen as an antibody comprising a heavy chain variable region(VH) having an amino acid sequence SEQ ID NO:2 (FIG. 1B) and a lightchain variable region (VL) having the amino acid sequence SEQ ID NO:1(FIG. 1A).
 52. The high affinity neutralizing immunoglobulin of claim49, wherein the immunoglobulin has a K_(a) of at least 10¹¹M⁻¹.
 53. Thehigh affinity neutralizing immunoglobulin of claim 50, wherein theimmunoglobulin has a K_(a) of at least 10¹¹ M⁻¹.
 54. The high affinityneutralizing immunoglobulin of claim 49, wherein the immunoglobulin hasa K_(a) is 10¹⁰ M⁻¹ or 10¹¹ M⁻¹.
 55. The high affinity neutralizingimmunoglobulin of claim 50, wherein the immunoglobulin has a K_(a) is10¹⁰ M⁻¹ or 10¹¹M⁻¹.
 56. The high affinity neutralizing immunoglobulinof claim 49, wherein the immunoglobulin neutralizes RSV as measured by amicroneutralization assay.
 57. The high affinity neutralizingimmunoglobulin of claim 56, wherein the immunoglobulin has an IC₅₀ inthe microneutralization assay that is less than the IC₅₀ of the IX-493antibody.
 58. The high affinity neutralizing immunoglobulin of claim 50,wherein the immunoglobulin neutralizes RSV as measured by amicroneutralization assay.
 59. The high affinity neutralizingimmunoglobulin of claim 58, wherein the immunoglobulin has an IC₅₀ inthe microneutralization assay that is less than the IC₅₀ of the IX-493antibody.
 60. The high affinity neutralizing immunoglobulin of claim 49,wherein the immunoglobulin is a tetrameric antibody, a Fab fragment, anF(ab)′₂, a heavy-light chain dimer, or a single chain structure.
 61. Thehigh affinity neutralizing immunoglobulin of claim 49, wherein theimmunoglobulin is a monoclonal antibody.
 62. The high affinityneutralizing immunoglobulin of claim 49, wherein the immunoglobulin is ahumanized antibody.
 63. The high affinity neutralizing immunoglobulin ofclaim 50, wherein the immunoglobulin is a tetrameric antibody, a Fabfragment, an F(ab)′₂, a heavy-light chain dimer, or a single chainstructure.
 64. The high affinity neutralizing immunoglobulin of claim50, wherein the immunoglobulin is a monoclonal antibody.
 65. The highaffinity neutralizing immunoglobulin of claim 50, wherein theimmunoglobulin is a humanized antibody.
 66. The high affinityneutralizing immunoglobulin of claim 50, wherein the immunoglobulinfurther comprises framework regions of a VL having the amino acidsequence of SEQ ID NO:1 and framework regions of a VH having the aminoacid of SEQ ID NO:2.
 67. A composition comprising the high affinityneutralizing immunoglobulin of claim 49, and a pharmacologicallyacceptable diluent or excipient.
 68. A composition comprising the highaffinity neutralizing immunoglobulin of claim 50, and apharmacologically acceptable diluent or excipient.
 69. A method forpreventing or treating a disease associated with RSV in a patient atrisk of such disease or afflicted with such disease, comprisingadministering the high affinity neutralizing immunoglobulin of claim 50to the patient.
 70. A process for producing a high affinity neutralizingimmunoglobulin that specifically binds to a RSV F antigen with a K_(a)of at least 10¹⁰ M⁻¹ as measured by surface plasmon resonance, theprocess comprising introducing into one or more of the CDRs of anexisting antibody one or more amino acid changes, wherein the existingantibody comprises the following CDRs and the one or more amino acidresidue changes are made at the boxed positions: a. a VL comprising thefollowing CDR sequences:

b. a VH comprising the following CDR sequences:


71. The process of claim 70, wherein the immunoglobulin binds to thesame epitope on the RSV F antigen as an antibody comprising a VHcomprising an amino acid sequence SEQ ID NO:2 (FIG. 1B) and a VLcomprising the amino acid sequence SEQ ID NO:1 (FIG. 1A).
 72. Theprocess of claim 70, wherein the immunoglobulin neutralizes RSV asmeasured by the microneutralization assay described in Example
 2. 73.The process of claim 72, wherein the immunoglobulin has an IC₅₀ in themicroneutralization assay that is less than the IC₅₀ of the IX-493antibody.
 74. The process of claim 70, wherein the immunoglobulin has aK_(a) of 10¹⁰ M⁻¹ or 10¹¹ M⁻¹.
 75. The process of claim 70, wherein theimmunoglobulin has a K_(a) of at least 10¹¹ M⁻¹.
 76. The process ofclaim 70, wherein the existing antibody further comprises frameworkregions of a VL having the amino acid sequence of SEQ ID NO:1 andframework regions of a VH having the amino acid of SEQ ID NO:2.